Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment

The storage of plant samples as well as sample preparation for extraction have a significant impact on the profile of metabolites, however, these factors are often overlooked during experiments on vegetables or fruit. It was hypothesized that parameters such as sample storage (freezing) and sample pre-treatment methods, including the comminution technique or applied enzyme inhibition methods, could significantly influence the extracted volatile metabolome. Significant changes were observed in the volatile profile of broccoli florets frozen in liquid nitrogen at −20 °C. Those differences were mostly related to the concentration of nitriles and aldehydes. Confocal microscopy indicated some tissue deterioration in the case of slow freezing (−20 °C), whereas the structure of tissue, frozen in liquid nitrogen, was practically intact. Myrosinase activity assay proved that the enzyme remains active after freezing. No pH deviation was noted after sample storage - this parameter did not influence the activity of enzymes. Tissue fragmentation and enzyme-inhibition techniques applied prior to the extraction influenced both the qualitative and quantitative composition of the volatile metabolome of broccoli.

The Impact of Domestic Cooking Methods on Myrosinase Stability, Glucosinolates and Their Hydrolysis Products in Different Cabbage (Brassica oleracea) Accessions

Glucosinolate hydrolysis products are responsible for the health-promoting properties of Brassica vegetables. The impact of domestic cooking on the myrosinase stability, glucosinolates and hydrolysis products in 18 cabbage accession was investigated. Cabbages were steamed, microwaved, and stir-fried before analysis. Cooking significantly affected myrosinase stability and glucosinolate concentrations within and between cabbage morphotypes. Myrosinase was most stable after stir-frying, with up to 65% residual activity. Steaming and microwaving resulted in over 90% loss of myrosinase activity in some accessions. Stir-frying resulted in the greatest decrease in glucosinolate concentration, resulting in up to 70% loss. Steamed cabbages retained the highest glucosinolates after cooking (up to 97%). The profile and abundance of glucosinolate hydrolysis products detected varied across all cooking methods studied. Cooking reduced the amounts of nitriles and epithionitriles formed compared to raw samples. Steaming led to a significant increase in the concentration of beneficial isothiocyanates present in the cabbage and a significantly lower level of nitriles compared to other samples. Microwaving led to a reduction in the concentrations of both nitriles and isothiocyanates when compared to other cooking methods and raw cabbage. The results obtained help provide information on the optimal cooking methods for cabbage, suggesting that steaming may be the best approach to maximising beneficial isothiocyanate production.

Influence of Cabbage (Brassica oleracea) Accession and Growing Conditions on Myrosinase Activity, Glucosinolates and Their Hydrolysis Products

Glucosinolates are secondary plant metabolites present in Brassica vegetables. The endogenous enzyme myrosinase is responsible for the hydrolysis of glucosinolates, yielding a variety of compounds, including health-promoting isothiocyanates. The influence of cabbage accession and growing conditions on myrosinase activity, glucosinolates (GSL) and their hydrolysis products (GHPs) of 18 gene-bank cabbage accessions was studied. Growing conditions, cabbage morphotype and accession all significantly affected myrosinase activity and concentration of glucosinolates and their hydrolysis products. In general, cabbages grown in the field with lower growth temperatures had significantly higher myrosinase activity than glasshouse samples. Profile and concentration of glucosinolates and their hydrolysis products differed across the accessions studied. Aliphatic glucosinolates accounted for more than 60 % of total glucosinolates in most of the samples assessed. Nitriles and epithionitriles were the most abundant hydrolysis products formed. The results obtained showed that consumption of raw cabbages might reduce the amount of beneficial hydrolysis products available to the consumer, as more nitriles were produced from hydrolysis compared to beneficial isothiocyanates. However, red and white cabbages contained high concentrations of glucoraphanin and its isothiocyanate, sulforaphane. This implies that careful selection of accessions with ample concentrations of certain glucosinolates can improve the health benefits derived from raw cabbage consumption.

Hijacking the Mustard-Oil Bomb: How a Glucosinolate-Sequestering Flea Beetle Copes With Plant Myrosinases

Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the β-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle’s energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants.

A specialist flea beetle manipulates and tolerates the activated chemical defense in its host plant

Glucosinolates, the characteristic secondary metabolites of Brassicales, are hydrolyzed upon herbivory by myrosinases to toxic and deterrent defense metabolites. The specialist flea beetle, Phyllotreta armoraciae, sequesters glucosinolates in the body despite myrosinase activity, but it is unknown whether plant myrosinase activity influences sequestration and how beetles prevent the hydrolysis of ingested glucosinolates. In feeding experiments performed with the myrosinase-deficient Arabidopsis thaliana tgg1tgg2 (tgg) mutant and the corresponding wild type, we found that plant myrosinases reduced the glucosinolate sequestration rate by up to 50% and hydrolyzed a fraction of ingested glucosinolates in adult beetles. Although these results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis, we observed no negative influence on beetle performance. To understand how P. armoraciae can avoid the hydrolysis of some ingested glucosinolates, we analyzed their fate directly after ingestion. P. armoraciae rapidly absorbed glucosinolates across the gut epithelium, a strategy that has been proposed to prevent hydrolysis in the gut lumen of sequestering insects. Moreover, beetle gut content suppressed in vitro myrosinase activity, and almost no myrosinase activity was detectable in the feces, which indicates that ingested myrosinases are inactivated in the beetle gut. In summary, we show that P. armoraciae uses several strategies to prevent the hydrolysis of ingested glucosinolates but can also tolerate the formation of glucosinolate hydrolysis products.

Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment

The storage of plant sample as well as sample preparation for extraction have a significant impact on the profile of metabolites. However, this parameter is often overlooked during experiments on plants. It was hypothesised that parameters such as sample storage (freezing) and sample pre-treatment methods, including the comminution technique or applied enzyme inhibition methods could significantly influence the extracted volatile metabolome.Significant changes were observed in the volatile profile of broccoli florets frozen in liquid nitrogen and at -20oC. Those differences were mostly related to the concentration of nitriles and aldehydes. Confocal microscopy indicated some tissue deterioration in the case of slow freezing (-20oC), whereas the structure of tissue, frozen in liquid nitrogen, was practically intact. Myrosinase activity assay proved that the enzyme remains active after freezing. No pH deviation was noted after sample storage - this parameter did not influence the activity of enzymes. Tissue fragmentation and enzyme-inhibition techniques applied prior to the extraction influenced both qualitative and quantitative composition of the volatile metabolome of broccoli.

Thermosonication for the Production of Sulforaphane Rich Broccoli Ingredients

A large proportion of broccoli biomass is lost during primary production, distribution, processing, and consumption. This biomass is rich in polyphenols and glucosinolates and can be used for the production of bioactive rich ingredients for food and nutraceutical applications. This study evaluated thermosonication (TS) (18 kHz, 0.6 W/g, 40–60 °C, 3–7 min) for the pre-treatment of broccoli florets to enhance enzymatic conversion of glucoraphanin into the bioactive sulforaphane. TS significantly increased sulforaphane yield, despite a decrease in myrosinase activity with increasing treatment intensity. The highest sulforaphane yield of ~2.9 times that of untreated broccoli was observed for broccoli thermosonicated for 7 min at 60 °C, which was 15.8% higher than the corresponding yield for thermal processing without sonication (TP) at the same condition. This was accompanied by increase in the residual level of glucoraphanin (~1.8 and 2.3 time respectively after TP and TS at 60 °C for 7 min compared to control samples) indicating that treatment-induced release of bound glucoraphanin from the cell wall matrix and improved accessibility could be at least partially responsible for the enhanced sulforaphane yield. The result indicates the potential of TS for the conversion of broccoli biomass into high sulforaphane broccoli-based ingredients.

Evaluating the Anti-Inflammatory and Antioxidant Effects of Broccoli Treated with High Hydrostatic Pressure in Cell Models

Isothiocyanates (ITCs) are important functional components of cruciferous vegetables. The principal isothiocyanate molecule in broccoli is sulforaphane (SFN), followed by erucin (ERN). They are sensitive to changes in temperature, especially high temperature environments where they are prone to degradation. The present study investigates the effects of high hydrostatic pressure on isothiocyanate content, myrosinase activity, and other functional components of broccoli, and evaluates its anti-inflammatory and antioxidant effects. Broccoli samples were treated with different pressures and for varying treatment times; 15 min at 400 MPa generated the highest amounts of isothiocyanates. The content of flavonoids and vitamin C were not affected by the high-pressure processing strategy, whereas total phenolic content (TPC) exhibited an increasing tendency with increasing pressure, indicating that high-pressure processing effectively prevents the loss of the heat-sensitive components and enhances the nutritional content. The activity of myrosinase (MYR) increased after high-pressure processing, indicating that the increase in isothiocyanate content is related to the stimulation of myrosinase activity by high-pressure processing. In other key enzymes, the ascorbate peroxidase (APX) activity was unaffected by high pressure, whereas peroxidase (POD) and polyphenol oxidase (PPO) activity exhibited a 1.54-fold increase after high-pressure processing, indicating that high pressures can effectively destroy oxidases and maintain food quality. With regards to efficacy evaluation, NO production was inhibited and the expression levels of inducible nitric oxide synthase (iNOS) and Cyclooxygenase-2 (COX-2) were decreased in broccoli treated with high pressures, whereas the cell viability remained unaffected. The efficacy was more significant when the concentration of SFN was 60 mg·mL−1. In addition, at 10 mg·mL−1 SFN, the reduced/oxidized glutathione (GSH/GSSG) ratio in inflammatory macrophages increased from 5.99 to 9.41. In conclusion, high-pressure processing can increase the isothiocyanate content in broccoli, and has anti-inflammatory and anti-oxidant effects in cell-based evaluation strategies, providing a potential treatment strategy for raw materials or additives used in healthy foods.

Effect of Ultrasound-Assisted Blanching on Myrosinase Activity and Sulforaphane Content in Broccoli Florets

Sulforaphane (SFN) is a health-promoting compound occurring in broccoli. It is formed by action of myrosinase in a two-step reaction that also yields undesirable compounds such as nitriles and isothionitriles. Different techniques affecting enzyme activity and tissue integrity were proposed to increase SFN content in the edible parts and discards of broccoli. Ultrasound processing is an emerging technology that produces these effects in foods, but has been poorly explored in broccoli so far. The aim of this work was to study the effect of ultrasound-assisted blanching on myrosinase activity and SFN content in broccoli florets. Myrosinase showed first-order inactivation kinetics in blanching at different temperatures with and without ultrasound processing. The inactivation rate was faster using ultrasound, with kinetic constants two orders of magnitude higher than without ultrasound. The activation energy (Ea) in traditional blanching (57.3 kJ mol−1) was higher than in ultrasound-assisted blanching (15.8 kJ mol−1). Accordingly, ultrasound accelerates myrosinase inactivation. The blanching time and temperature significantly affected myrosinase activity and SFN content. At 60 °C and 4 min of ultrasound-assisted blanching, myrosinase activity was minimum and SFN content was the highest. These findings may help to design SFN enrichment processes and will contribute to the valorization of agro-industrial wastes.